ES2199305T3 - USE OF LACTOBACILLUS REUTERI TO INHIBIT CRYPTOSPORIDIOSIS IN MAMMALS. - Google Patents

Abstract

CRYPTOSPORIDIUM PARVUM (THE CAUSE OF CRYPTOSPORIDIOSIS) HAS BECOME ONE OF THE MOST COMMON ENTEROPATHOGENS THAT PRODUCE DIARRHEA WORLDWIDE. SYMPTOMS ASSOCIATED WITH CRYPTOSPORIDIOSIS ARE VERY DEBILITATING, ESPECIALLY IN AN IMMUNOCOMPROMITED SUBJECT (FOR EXAMPLE A PATIENT WITH AIDS). CLINICAL CHARACTERISTICS INCLUDE SERIOUS AND CHRONIC DIARRHEA, ABDOMINAL CALAMBRES, FATIGUE, WEIGHT LOSS, ETC. THAT LEAD TO AN INCREASE IN HEALTH COSTS AND MORTALITY. A PROCEDURE FOR INHIBITING THE GRAVITY OF AN INFECTION BY CRYPTOSPORIDIUM PARVUM IS DESCRIBED IN THE DESCRIPTIVE MEMORY THROUGH THE ENTERICABLE ADMINISTRATION OF A THERAPEUTICALLY EFFECTIVE AMOUNT OF REUTERIALLY LACTOBACILLUS.

Description

Use of Lactobacillus reuteri to inhibit cryptosporidiosis in mammals.

Field of the Invention

This invention relates to the use of Lactobacillus reuteri for the inhibition of stages of diseases associated with Cryptosporidium parvum .

Background of the invention

Cryptosporidiosis is caused by the protozoan parasite Cryptosporidium parvum (C. parvum) . Historically, C. parvum has been recognized as a cause of diarrhea and mortality in young animals with an underdeveloped immune system. It was not until the outbreak of acquired immunodeficiency syndrome (AIDS) that C. parvum was considered a major problem in humans (Petersen, `` Cryptosporidiosis in patients infected with the human immunodeficiency virus, '' Clinical Infectious Diseases , 15: 903 -909, 1992). Increased knowledge and improved diagnostic techniques indicate that C. parvum may now be one of the three most important enteropathogens that causes diarrhea disease worldwide (Laughten, et al., `` Summary of the workshop on future directions in discovery and development of therapeutic agents for opportunistic infections associated with AIDS '', The Journal of Infectious Diseases , 164: 244-251, 1991). Outbreaks have been reported in pediatric patients in hospitals (Navarrete, et al., `` An outbreak of Cryptosporidium diarrhea in a pediatric hospital, '' The Pediatric Infectious Disease Journal , 10: 248-250, 1991) and primary care centers (Anonymous, `` Cryptosporidiosis among children attending day-care centers - Georgia, Pennsylvania, Michigan, California, New Mexico, '' Morbid. Mortal. 33: 599-601, 1984). Even more recently, a large burst through the water in Milwaukee, Wisconsin, affected more than 400,000 people (MacKenzie, et al., `` A massive outbreak in Milwaukee of Cryptosporidium infection transmitted thorugh the public water supply, '' New England Journal of Medicine , 331: 161-167, 1984).

C. parvum infection is acquired through the ingestion of oocysts from the feces of infected animals or humans that remain in the environment. During the life cycle of C. parvum , numerous oocysts can be produced that can be excreted in the environment or remain inside the host to cause self-infection (Fayer, and Unger, `` Cryptosporidium spp. And cryptosporidiosis, '' Microbiological Reviews , 50 : 458-483, 1986). The oocysts are surprisingly resistant to most commonly used disinfectants, and it is likely that routine chlorination of drinking water has no effect on their viability (Current, `` The biology of Cryptosporidium , '' ASM News}, 54: 805-811, 1988). This environmentally resistant oocyst helps the transmission of organisms through either contamination of the drinking water supply or by contact between individuals. Some research suggests that only 30 oocysts are needed to infect an individual, but in the case of an immunosuppressed individual, only one could suffice. This could be based on the fact that only 8 to 10 oocysts were found per 100 liters of water in the recent outbreak in Milwaukee (Schwartz, et al., `` Biliary cryptosporidiosis in HIV + patients after a waterbourne outbreak in Milwaukee, '' Gastroenterology , 106 (4): A770 (summary), 1984). Interestingly, the U.S. government recently issued the following notice for tap water: `` Federal health officials have warned the nation that the daily threat of cryptosporidiosis is so widespread that everyone with a weakened immune system may wish to avoid drinking water straight from the tap. The U.S. Environmental Protection Agency and the Centers for Disease Control and Prevention indicate that people with severely weakened immune systems should take precautions such as boiling water, installing filters or using bottled water. Those at risk are those with AIDS or HIV; cancer patients; transplant patients; people with a genetically weakened immune system, and malnourished children, '' [The Columbus Dispatch Saturday, June 17, 1985 page 4). To date, no therapy has proven clinically effective.

Although cryptosporidiosis can be problematic for anyone, the most notable impact has been among patients with the human immunodeficiency virus (HIV). Clinical signs include chronic diarrhea (similar to cholera), abdominal cramps, tiredness, weight loss, etc. In immunocompetent people, the onset of this disease is explosive but is generally self-limiting after approximately two weeks. However, this state is very debilitating for the immunosuppressed individual and constitutes a threat to life. Recently, Blanshard and Gazzard (`` Natural history and prognosis of diarrhoea of unknown cause in patients with acquired immunodeficiency syndrome (AIDS), '' Gut , 36: 283-286, 1995) have shown that diarrhea associated with C. parvum in AIDS patients correlates with a significantly shorter median survival time. In their study, the median survival time for patients with `` pathogen-negative '' diarrhea was 48.7 months, which was similar to that of control patients without diarrhea; however, the median survival time was significantly longer than that of equivalent patients with a gastrointestinal pathogen (9.6 months). Of those patients with a positive diarrhea for the pathogen, it was determined that the cause was C. parvum in approximately 40% of cases. Others have reported a higher mortality rate in AIDS patients with cryptosporidiosis compared to non-infected AIDS patients (Gilson, et al., `` Impact of a community-wide outbreak of cryptosporidiosis on patients with AIDS, '' Tenth International Conference of AIDS 2:24 (summary), 1994).

The true prevalence of cryptosporidiosis is unknown. It has been estimated that 10 to 20 percent of AIDS patients in the United States have developed chronic diarrhea of C. parvum , while the incidence is probably higher in developing countries where contaminated water supplies constitute a big concern. The reported incidence rates underestimate the existence of C. Parvum infection,} because in the past its pathogenicity in humans was not recognized and because of the relative difficulty in identifying it.

Although numerous therapies have been tried recently, none of them seems to improve this debilitating state. A method to reduce intestinal infestation by C. parvum through a daily enteral supplement with probiotics is described. Probiotics have been defined as a live microbial nutritional supplement that affects the host beneficially because it improves intestinal microbial balance (Fuller, `` Probiotics in man and animals, '' Journal of Applied Bacteriology , 66: 365-378, 1989 ). Some researchers believe that this normalization of the intestinal microbiota will confer the following benefits: (a) protection against pathogens through competitive exclusion (also called colonization resistance); (b) the provision of certain nutrients and enzymatic / detoxifying reactions; (c) the involvement in tissue morphogenesis and peristaltic activity; and (d) interaction with the host's immune and endocrine systems. (Speck, et al., `` Lactobacillus reuteri in food supplementation, '' Food Technology , July: 90-94, 1983). Examples of probiotic organisms include several lactic acid bacteria such as lactobacilli, streptococci and bifidobacteria.

The most frequent lactobacilli found in the intestine in healthy people are Lactobacillus reuteri (L. reuteri) and Lactobacillus acidophilus (L. acidophilus). L. reuteri is a ubiquitous organism in men and animals. Of the intestinal lactic acid bacteria (BAL), L. reuteri is considered one of the most dominant species. Due to the inability of the microbiologist to differentiate L. reuteri from Lactobacillus fermentum (L. fermentum) in the past, many researchers believe that a high percentage of BALs classified as L. fermentum in the oldest literature are actually strains of L. reuteri .

L. reuteri is a species of heterofermentation typical of Lactobacillus . Like other lactobacilli, L. reuteri produces acidic metabolic end products (acetate and lactate) that have considerable antimicrobial activity. It has recently been discovered that glycerol metabolism by L. reuteri can result in the excretion of a metabolic intermediate, 3-hydroxypropionaldehyde (reuterine; Axelsson, `` Production of a broad spectrum antimicrobial substance by Lactobacillus reuteri , '' Microbial Ecology in Health and Disease , 2: 131-138, 1989). This compound has been shown to have an antimicrobial activity against a variety of organisms, including Gram-positive and Gram-negative bacteria, yeasts, molds and protozoa (Chung, et al., `` In vitro studies on reuterin synthesis by Lactobacillus reuteri , '' Microbial Ecology in Health and Disease , 2: 137-144, 1989). It is suspected that reuterine activity contributes to the survival of L. reuteri within the gastrointestinal ecosystem.

Likewise, L. acidophilus is a normal inhabitant of the human gastrointestinal tract. L. acidophilus is a species of homofermentation, which ferments mostly hexose sugars, predominantly providing lactic acid (85-95%). The use of L. acidophilus dates from the early twentieth century.

In accordance with one aspect of the present invention, there is provided a medicament for the inhibition of infection of a mammal by Cryptosporidium parvum using entectorally administered Lactobacillus reuteri in an amount that is therapeutically effective to inhibit said infection.

A medicament for inhibiting infection of a mammal with Cryptosporidium parvum using Lactobacillus reuteri enterally is provided in accordance with another aspect of the present invention in an amount that is therapeutically effective for inhibiting such infection, as evidenced by a reduction. of the oocyst shedding of Cryptosporidium parvum in feces.

According to another aspect of the present invention, a medicament is provided for the inhibition of infection of the intestine of a mammal by oocysts of Cryptosporidium parvum using entectorally administered Lactobacillus reuteri in an amount that is therapeutically effective to inhibit said infection.

In accordance with another aspect of the present invention, a medicament for the inhibition of infection of the intestine of a mammal by the oocysts of Cryptosporidium parvum using Lactobacillus reuteri administered enterally in an amount that is therapeutically effective is provided, is evidenced by a reduction of the oocyst shedding of Cryptosporidium parvum in feces.

A medicament for increasing resistance to Cryptosporidium parvum infection in an immunocompromised mammal is provided according to another aspect of the present invention using enterally administered Lactobacillus reuteri in an amount that is therapeutically effective for the inhibition of said infection.

A medicament for increasing resistance to infection with Cryptosporidium parvum in an immunocompromised mammal is provided in accordance with another aspect of the present invention using enterally administered Lactobacillus reuteri in an amount that is therapeutically effective to inhibit such infection, evidence by a reduction of the oocyst shedding of Cryptosporidium parvum in feces.

Brief description of the drawings

Figure 1 is a graphic representation of the Results of Experiment 1.

Detailed description of the invention

Two experiments were carried out to determine the effect of L. reuteri or L. acidophilus on C. parvum infection in a murine model of AIDS.

Clinical models

Cryptosporidiosis is the only opportunistic infection of AIDS patients for which there is no clinically effective therapy. Generally, it is recognized that the best described model is that of the mouse. Several `` types '' of mouse models have been described, including that of the neonatal mouse devoid of the BALB / c immune system. This mouse develops a persistent infection; however, these mice become more resistant to infection after 42 days of age (Heine, et al., `` Persistent Cryptosporidium infection in congenitally athymic (nude) mice, '' Infection and immunity 43 (3): 856 -859, 1984). In addition, models have been described in mice suffering from severe combined immunodeficiency (IDCG) and mice receiving anti-T-cell antibodies (Harp, et al., `` Resistance of severe combined immunodeficient mice to infection with Cryptosporidium parvum : the importance of intestinal microflora, '' Infection and immunity , 60 (9): 3509-3512, 1992; Ungar, et al., `` New mouse models for chronic Cryptosporidium infection in immunodeficient hosts, '' Infection and immunity 58 (4): 961- 969, 1990). A model of special interest is the mouse infected with SIDAM (murine acquired immunodeficiency syndrome). In this model, mice are infected with SIDAM leukemic retroviruses in an attempt to mimic the evolution of the disease that occurs in human AIDS. The benefits of this model include a similar reduction in T-cell response and progressive splenomegaly and lymphadenopathy such as in HIV / AIDS, in addition to a relatively rapid development of the disease (Alak, et al., `` Alcohol and murine acquired immunodeficiency syndrome suppression of resistance to Cryptosporidium parvum infection during modulation of cytokine production, '' Alcoholism: Clinical and Experimental Research , 17 (3): 539-544, 1993). It has been suggested that this model provides means for the study of resistance mechanisms to C. parvum infection and methods for the treatment of cryptosporidiosis (Darban, et al., `` Cryptosporidiosis facilitated by murine retroviral infection with MO-LP5, ''' The Journal of Infectious Diseases , 164: 741-743, 1991).

A summary presented by Famularo et al., (`` Effect of lactobacilli on Cryptosporidium parvum infection in man and animals, '' Microbial Ecology in Health and Disease , 8 (1): III, 1985) suggested that a mixture of numerous organisms Lactobacilli ( S. thermophilus, L. acidophilus, L. bulgaricus, L. casei, L. plantarum and a mixture of bifidobacteria) was effective in improving the symptoms associated with cryptosporidiosis. The present invention clearly documents the ability of a single organism to inhibit the infectivity of C. parvum .

Experiment 1 (Exp. one)

The objective of this study was to determine whether a pretreatment with L. reuteri could prevent or suppress C. parvum infection in female C57BL / 6 mice immunosuppressed by inoculation with MO-LP5 (lymphoproliferative bone marrow 5)

Materials and methods Mice

Female C57BL / 6 mice were purchased (3 to 4 weeks old) from Harian Sprague Dawley, Inc. (Indianapolis, Indiana). The mice were housed in a unit micro-insulator with a filtration system air. Five mice were housed per cage to reduce the aggressiveness. The accommodation facility was kept at a temperature of 20 to 22 ° C and a relative humidity of 60 to 80%. I know exposed the animals to a light / dark cycle of 12-12 hours and they were allowed to consume water and feed for mice (Purina Company, St. Louis, Missouri) ad libitum The mice were kept in the facility according to the standardized standards set by the Committee for the Care of Animals of Tuskegee University (Tuskegee, Alabama) for the Health and welfare of animals.

Inoculation of mice with MO-LP5

Mice were inoculated intraperitoneally (IP) with 0.30 milliliters (ml) of murine leukemic virus MO-LP5 (VLMu) that had an acotropic title of 4.5 log_10 PFU / ml (plaque forming units / mL) in a line of XC cells The strain of VLMu was provided by Dr. Ronald R. Watson (University of Arizona School of Medicine, Tucson, Arizona, original source: Dr. Robert Yetter, Center for Biological Evaluation and Research, FDA, Bethesda, Maryland).

C. parvum inoculum

Inoculated sterilized oocysts of C. parvum were provided by Dr. James A. Harp (National Center for Animal Diseases, Ames, Iowa). C. parvum oocysts were purified from experimentally infected calves as described above (Harp, et al., `` Resistance of severe combined immunodeficient mice to infection with Cryptosporidium parvum ; the importance of intestinal microflora '', Infection and Immunity , 60 (9): 3509-3512, 1992).

Preparation of probiotic bacteria

Two isolates of mouse L. reuteri (one isolated from the stomach and one from the small intestine, strains 4000 and 4020, respectively) were obtained from BioGaia Biologics, Inc., (Raleigh, North Carolina). The L. reuteri strain was supplied as frozen preparations in 0.1% peptone water for inoculation of the mice. The L. reuteri strains were developed separately and then the same number of colony forming units (CFU) of each strain were joined to give a final concentration of 5 x 10 8 CFU / ml. It is generally recognized that the ability to colonize the mucosa by lactobacilli (e.g., L. reuteri ) is specific to the host (Lin and Savage, `` Host specificity of the colonization of murine gastric epithelium by lactobacilli, '' FEMS Microbiology Letters 24: 67-71, 1984). In order for a probiotic to compete with pathogens for adhesion receptors, it must be able to colonize the intestine. Therefore, L. reuteri isolated from mice was used in the studies.

Experimental design

In total, 40 mice were randomly assigned C57BL / 8 females (10 mice per group) to one of four treatments (A, B, C or D; Table 1) after they had been immunosuppressed for 4 months by prior inoculation with MO-LP5.

TABLE 1

Description of the treatment groups for Exp. 1 Treatment Group 1 MO-LP5 2 L. reuteri3 C. parvum 4 TO + - - B + - + C + + - D + + + 1 ten mice per group (5 mice per cage). 2 immunosuppressed for 4 months per inoculation with MO-LP5. 3 L. reuteri force fed daily (1.0 x 10 8 CFU per mouse) during the sizing phase and the phase experimental. 4 infected with C. parvum (8.5 x 10 8 oocysts per mouse) 11 days after supplementation with L. reuteri .

The experiment was divided into a sizing phase and an experimental phase. The sizing phase lasted 10 days during which 20 mice (groups C and D) were force fed with L. reuteri (1 x 10 8 CFU in 0.2 ml) daily. The remaining 20 mice (groups A and B) were force fed daily with phosphate buffered saline (STF). Mice that received L. reuteri or STF during the sizing phase were treated similarly during the experimental phase, while the animals had access to food and water ad libitum. During the sizing phase, fecal samples were collected on days 0 (baseline), 4 and 7 for the total lactobacillus and L. reuteri count. On day 10, faeces were collected from all mice for the detection of oocyst shedding of C. parvum and the total lactobacillus and L. reuteri count. The experimental phase was started on day 11 of the study, during which the mice were exposed to 6.5 x 10 6 oocysts of C. parvum in 0.2 ml of sterilized STF. Previously, an oocyst concentration of 2.0 x 10 5 had been shown to enhance growth and reduce variability in the stripping of parasites from the intestinal tract of animals from the same experimental groups (Darban, et al., ` `` Cryptosporidiosis facilitated by murine retroviral infection with MO-LP5, '' The Journal of Infectious Diseases , 164: 741-745, 1991). Fecal samples were collected on days 17 and 25 for the count of L. reuteri , total Lactobacillus and C. parvum . On day 26, the mice were sacrificed by ether inhalation. Then, 1 to 2 centimeters (cm) of the proximal stomach, distal ileum and colon of each mouse were removed, and fixed in 10% formalin. Daily food and water intake, initial and final body weight, and diarrhea development were recorded.

Lactobacillus sampling

Fresh fecal samples (3 to 4 feces) were weighed and placed in small sterilized vials (resistant to -70 ° C). The samples were quickly frozen using alcohol and carbonic snow and stored at -70 ° C until transport. The samples were transported on carbonic snow with delivery the next day to BioGala Biologics, Inc, for the Lactobacillus spp. Totals and L. reuteri . L. reuteri and Lactobacillus spp. Total using standard techniques for anaerobic microbes and / or techniques developed by BioGala Biologics, Inc., as described above (Wolf, et al., `` Safety and tolerance of Lactobacillus reuteri in healthy adult male subjects, '' Microbiological Ecology in Health and Diseases 8: 41-50, 1995). Lactobacillus spp. Total and L. reuteri fecal were recorded as CFU / gram (g) of wet feces. In this experiment, colonization has been defined as more than 5.0 x 10 3 CFU / g of wet feces.

Dessert of parasites of C. parvum

Goodgame et al., (`` Intensity of infection in AIDS-associated cryptosporidiosis '', The Journal of Infectious Diseases , 167: 704-709, 1983) found a significant correlation between fecal excretion of oocysts and the numbers of C organisms Parvum observed in biopsies of the small intestine. They suggested that there is a threshold of infection intensity below which symptoms do not appear and that a reduction in oocyst excretion, even in the absence of total eradication, may be a valid measure of the efficacy of a drug. In this experiment, C. parvum oocysts stripped in feces were estimated as oocysts / g of feces as previously described (Alak, et al., `` Alcohol and murine acquired immunodeficiency syndrome suppression of resistance to Cryptosporidium parvum infection during modulation of cytokine production, '' Alcoholism: Clinical and Experimental Research , 17 (3): 539-544, 1993). In summary, 3 to 4 feces of each mouse were collected in sterile tubes of microcentrifuge previously weighed. The weight of each stool sample was determined by subtracting the weight of each microcentrifuge tube from the total weight of the tubes plus the fecal content. One ml of 10% formalin buffered solution was added to each microcentrifuge tube and the samples were stored at room temperature or at 4 ° C until analysis. First, the faecal samples were mixed using applicator sticks, then stirred thoroughly. Appropriate dilutions were made in STF, filtered through millipore filters with a passage light of 0.45 micrometers (µm) (Micron Separation Inc., Westboro, Massachusetts) in a housing chamber (Gelman Sciences Inc., Ann Arbor, Michigan) using 5 ml syringes. The lower ends of the chamber were wrapped with PARAFILM tape (parafilm is a registered trademark of American Can Co.) and 150 microliters (µl) of a 1: 8 dilution of Cryptosporidium detection reagent (Meridian Diagnostics, Inc ., Cincinnati, Ohio) diluted in STF to each chamber and allowed to incubate at room temperature for 1 hour protected from light. The PARAFILM tape was removed and the chambers were washed 3 times with STF using 5 ml syringes. The lower ends of the chambers were sealed with PARAFILM tape, then 150 µl of counterstain reagent, diluted 1:10 in STF, was added to each chamber, then incubated and then washed. The filters containing the oocysts retained on the upper surfaces of the filters were mounted on glass plates (Meridian Diagnostics, Inc.). One or 2 drops of buffered glycerin, diluted 1: 9 in STF, was added. Covers were applied to cover them and the edges were sealed with transparent nail polish (Pavlon LTD, Nyack, New York). The C. parvum oocysts showed a characteristic staining of apple green on the walls when they were observed in a Leitz fluorescent microscope (Leica Inc., Chicago, Illinois) using a 40x objective lens. Taking into account the dilution factor of the sample, the total weight and volume of the feces and the count of oocysts per filter, the number of oocysts stripped per gram of feces was estimated. For the count of parasitic colonization of the intestinal epithelium, 1 to 2 cm of distal ileum of each mouse was surgically removed and rinsed in PBS. Each intestinal section was stained histologically by the conventional hematoxylin / eosin method. The average number of oocysts colonized per centimeter of the intestine was counted using a hemocytometer under a phase contrast microscope with a 40x objective lens. Infectivity scores for histological sections were determined as the number of oocysts per cm of organ.

Statistic analysis

Since mice were handled in cages of 5 animals each, the cages were placed within the groups of treatment. The result was a Variance Analysis model (ANOVA) placed with a main treatment effect (A, B, C, D) and a placed effect (a cage in a given treatment). I know examined all the data to assess the assumption of a normal waste distribution according to the ANOVA model placed, examining waste by testing Shapiro-Wilk. The data that fit the Assumption of normality were analyzed using the ANOVA model placed. Data that did not fit the assumption of normality were ordered and the resulting order was analyzed using The ANOVA placed. For significant treatment effects, the treatment media were compared through the HSD test of Tukey The means of treatment were significant if they differed from each other at a probability level of 5%.

Results - Exp. 1 Body weights

The mice in Groups A, B and C had a small reduction in body weight unlike mice in Group D, whose body weight remained unchanged until end of the study The mice in Group A had the weight lower body of all groups at baseline (21,45 compared with 23.08, 23.66 and 23.22 g / mouse for Groups A, B, C and D, respectively) that can be correlated with their weights lower end body (p <0.05) (21.13, 22.55, 22.30 and 23.28 g / mouse for Groups A, B, C and D, respectively). The mice in Group D had the highest body weights though this parameter was not different (P> 0.05) for mice of Groups B and C.

       \ newpage
    

Food and water consumption

Since 5 animals were housed for each cage, individual intakes could not be determined, therefore these data were tabulated for descriptive purposes only. The food intake was similar among all the Treatment Groups (2.80, 2.98, 2.97 and 3.19 g / mouse / day for Groups A, B, C and D, respectively). Mice supplemented with L. reuteri (Groups C and D) had higher water consumption (4.95 and 4.75 ml / mouse / day, respectively) than Groups A and B (3.83 and 3.98 ml / mouse / day, respectively) who received STF.

Dispossession of C. parvum

No oocysts of C. parvum were detected in the feces of the mice (Groups A and C) not exposed to C. parvum . However, mice exposed to parasites (Groups B and D) developed persistent cryptosporidiosis
(Table 2). Infection with C. parvum without supplement of L. reuteri (Group B) increased (p <0.05) oocyst shedding at 7 and 14 days after infection. Although there were no differences (p> 0.05) in the oocyst shedding between Groups B and D at 7 days after infection, the offal was reduced (p <0.05) at 14 days after exposure in the mice receiving supplements with L. reuteri (Group D). There was absence of infection with the Cryptosporidium parasite in the intestinal epithelium (specifically the distal ileum) of Group D mice. In addition, no parasites were detected in the intestinal hairs of uninfected mice (Groups A and C). However, significant parasite loads (p <0.05) were detected in the intestines of mice (Group B) infected with C. parvum alone. Contrary to colonization of the distal ileum, no parasites of C. parvum were observed in the tissues of the stomach or colon of exposed mice.

TABLE 2

Effects of feeding with L. reuteri supplements on oocyst shedding (oocysts x 10 3 g ± SEM) in the feces and colonization of the epithelium of the distal ileum (oocysts / cm of intestine ± EEM) of C578BL / 6 mice immunosuppressed by prior inoculation with MO-LP5 and exposed (+/-) to C. parvum Days after feeding with L. Reuteri Group 1 Day 10 2 Day 17 3 Day 25 4 Ileum TO 0.00 ± 0.00 0.00 ± 0.00 b 0.00 ± 0.00 c 0.00 ± 0.00 b B 0.00 ± 0.00 1.58 ± 0.24 a 9.19 ± 4.29 a 4.00 ± 1.13 a C 0.00 ± 0.00 0.00 ± 0.00 b 0.00 ± 0.00 c 0.00 ± 0.00 b D 0.00 ± 0.00 1.34 ± 0.33 a 0.46 ± 0.13 b 0.00 ± 0.00 b 1 Ten mice per group (5 mice per cage). 2 Corresponds to a baseline sample taken just before exposure to C. parvum 3 Corresponds to day 7 after exposure to C. parvum 4 Corresponds to day 14 after exposure to C. parvum ^ a, b, c} Values within the same column with different superscript letters are different (p < 0.05).

Colonization by Lactobacillus

Faecal levels of Lactobacillus spp. Totals were similar among all treatments throughout the study (Table 3). Only statistically different levels were found on day 17 (Group B> Group A). The level of L. reuteri in the stool was similar (> 0.05) among all treatments on day 0 (baseline). On day 4, treatment groups C and D had higher levels of L. reuteri (p <0.05) and tended to have higher levels on day 7. However, on days 10, 17 and 25, all the treatment groups, with the exception of Group B consistently stripped higher levels of L. reuteri in feces. The percentage of mice colonized by L. reuteri is shown in Figure 1, which shows the effect of L. reuteri or STF on the percentage of mice colonized by L. reuteri (colonization is defined as more than 5.0 x 10 ^ {3} CFU of L. reuteri / g wet feces). In general, treatment groups fed with L. reuteri supplement had a higher percentage of animals that were colonized by the organism compared to untreated groups. In addition, on days 10, 17 and 25, mice in Group D had a higher percentage (p <0.05) of colonization than mice in Group B.

TABLE 3

Effects of feeding with L. reuteri supplements on the fecal level of Lactobacillus spp. totals and L. reuteri (log 10 CFU / gEEM) of C57BL / 6 mice immunosuppressed by prior inoculation with MO-LP5 and exposed (+/-) to C. parvum Total lactobacilli - days after feeding with L. reuteri Group 1 Day 1 Day 4 Day 7 Day 10 Day 17 Day TO 8.90 ± 0.11 9.11 ± 0.12 9.07 ± 0.12 9.25 ± 0.06 8.49 ± 0.17 b 9.15 ± B 8.83 ± 0.11 8.82 ± 0.06 * 9.04 ± 0.07 * 9.00 ± 0.08 9.18 ± 0.10 a ** 9.07 C 8.71 ± 0.11 9.77 ± 0.12 9.33 ± 0.08 9.22 ± 0.15 ** 9.01 ± 0.10 ^ a, b ** 9.07 D 8.60 ± 0.08 9.70 ± 0.11 9.35 ± 0.15 9.12 ± 0.20 8.93 ± 0.18 a, b 8.98 ± L. reuteri TO 4.85 ± 0.48 4.67 ± 0.51 b 5.29 ± 0.60 a, b 6.12 ± 0.68 6.88 ± 0.24 7.15 ± B 4.25 ± 0.28 4.65 ± 0.39 a * 5.17 ± 0.47 b * 5.29 ± 0.65 5.76 ± 0.69 ** 5.01 C 4.33 ± 0.28 6.79 ± 0.60 a 6.75 ± pm 0.39 a, b 6.20 ± 0.19 ** 6.66 ± 0.51 ** 7.64 D 5.13 ± 0.39 7.48 ± 0.45 a 6.88 ± 0.49 a 7.97 ± 0.23 7.26 ± 0.16 8.16 ± pm 1 Ten mice per group (5 mice per cage), unless stated otherwise: * = 9; ** = 8. ^ a, b} Values within it column with different superscript letters are different (p < 0.05).

Discussion Exp. 1

Infection of mice with MO-LP5 induced murine AIDS syndrome without any latent phase. This syndrome had many similarities with human AIDS. Symptoms included progressive splenomegaly, lymphadenopathy and an increased susceptibility to C. parvum infection. Unlike human AIDS, which is associated with weight loss and sometimes with a diarrhea that threatens the patient's life, murine AIDS was not associated with significant weight loss or diarrhea.

Treatment of mice with L. reuteri increased (p <0.05) the intestinal population of L. reuteri (Groups C and D) compared to mice that did not receive a supplement of L. reuteri (Groups A and B) on day 4 after feeding with L. reuteri . There was an inverse relationship between the population of L. reuteri and the clearance of parasites with C. parvum from the intestinal tract in treatment groups B and D. Therefore, mice infected with C. parvum and fed concomitantly with L. reuteri ( Group D) clarified (p <0.05) parasite loads (Table 2) compared to those infected by C. parvum alone (Group B). At 14 days after exposure to Cryptosporidium , no parasites were detected in the epithelium of the intestinal tract of these mice (Group D). In contrast, control mice exposed to Cryptosporidium could not clear up the infection 14 days after exposure. This demonstrates a prophylactic role, hitherto unrecognized, of L. reuteri in the inhibition of the growth of C. parvum in the intestinal mucosa. The mechanisms by which L. reuteri inhibits the proliferation of C. parvum are unknown. It is speculated that L. reuteri can inhibit the proliferation of other bacteria and other parasites in the gut microbiota through competition for binding sites or through the secretion of products that inhibit other organisms. This study provides evidence that L. reuteri is beneficial for the inhibition of opportunistic infections such as C. parvum , especially in immunocompromised individuals.

Experiment 2 (Exp. two)

A second experiment was carried out to determine whether pretreatment with L. reuteri or L. acidophilus could prevent or inhibit C. parvum infection in mice infected with SIDAM.

Materials and methods Mice

They got and took care of mice of the same so in Exp. 1, except that the mice had a notch in the ear to identify each animal.

Inoculation of mice with MO-LP5

The same as Exp. 1.

Cryptosporidium inoculums

The same as Exp. 1

Preparation of probiotic bacteria

L. reuteri was prepared as explained in Exp. 1. A human isolate of L. acidophilus (NCFM strain) was obtained from BioGala Biologics, Inc. L. acidophilus was prepared with a final concentration of 5 x 10 8 } CFU / mL. Probiotic ingredients were stored frozen in 0.1% peptone water until use.

Experimental design

In this experiment, 60 female C57BL / 6 mice (15 per group) were randomly assigned to one of four treatments (E, F, G and H; Table 4). Again, the study is divided into a sizing phase and another experimental phase.

TABLE 4

Description of the treatment groups for the Exp. 2 Treatment Group 1 MO-LP5 2 L. reuteri3 L. acidophilus 4 C. parvum 5 TO + - - - B + - - + C + + - + D + - + + 1 Fifteen mice per group (5 mice per cage). 2 Immunosuppressed for 4 months by inoculation with MO-LP5. 3 L. reuteri force fed daily (1.0 x 10 8 CFU per mouse) during the sizing phase and the phase experimental. 4 L. force-fed acidophilus daily (1.0 x 10 8 CFU per mouse) during the sizing phase and the phase experimental. 4 Infected with C. parvum (8.5 x 10 8 oocysts per mouse) 14 days after probiotic supplementation.

During the sizing phase (a duration of 13 days), 30 mice (Groups E and F) were force fed with 0.1% peptone water (0.2 ml), 15 mice (Group G) were fed by force with L. reuteri (1 x 10 6 CFU in 0.2 ml) and 15 mice (Group H) were force fed daily orally with L. acidophilus
(1 x 10 6 CFU in 0.2 ml). Mice continued to be fed by force daily orally with their respective treatments during the experiment. During the sizing phase, fecal samples were taken on day 1 (baseline) and on day 8 for the Lactobacillus spp. Totals and L. reuteri . Attempts were made to determine the faecal levels of L. acidophilus with some samples of Group H; however, the method was too cumbersome and variable, so the L acidophilus analysis was not completed. On day 13, faecal samples were collected from all mice for the detection of C. parvum offal and the Lactobacillus spp. Totals and L. reuteri . The experimental phase began on day 14 during which the animals (Groups F, G and H) were exposed to C. parvum (1.4 x 10 5 oocysts). Fecal samples were collected on days 21 and 28 (7 and 14 days after exposure) for the count of C. parvum , Lactobacillus spp. Totals and L. reuteri . On day 28, the mice were sacrificed by ether inhalation. Then, 1 to 2 cm of the proximal stomach, distal ileum and colon of each mouse were removed for the detection of C. parvum in the intestine epithelium. Documentation of daily food and water intake, and diarrhea were recorded. In addition, initial and final body weights were recorded.

Lactobacillus sampling

Same as Exp. 1. Due to variations in the detection limits of Exp. 2, colonization was defined as more than 2.7 x 10 4 CFU of L. reuteri / g stool wet

       \ newpage
    

Dessert of parasites of C. parvum

The same as Exp. 1, except that infectivity scores for histological sections of the Distal ileum were determined as number of oocysts per 25 hairs Intestinal

Statistical analysis

The same as Exp. 1. The animals individually, but in cages of 5 animals each one, therefore, the cages were placed within a group of treatment. This results in a variance analysis model placed with a main treatment effect (E, F, G and H) and a nested effect (cage in treatment). Methods statistics were similar to those described for Exp. 1. Of again, the results were considered statistically significant if the level of significance was less than 5 per hundred.

Results - Exp. 2 Body weights

The initial body weights (21.80, 22.82, 22.03 and 22.48 g / mouse for Groups E, F, G and H, respectively) and finals (22,18, 23,01, 22,28 and 23,07 g / mouse for Groups E, F, G and H, respectively) were similar (p <0.05) among all the treatments In general, the mice showed a slight increase in body weight for the duration of the study.

Food and water consumption

Food consumption (3.33, 3.40, 3.33 and 3.40 g / mouse / day for Groups E, F, G and H, respectively) and water (3.92, 3.97, 3.98 and 3.96 ml / mouse / day for Groups E, F, G and H, respectively) were similar between treatments.

Dispossession of C. parvum

No oocysts of C. parvum were detected in the feces of the mice (group E) not exposed to C. parvum . As in Exp. 1, exposed animals developed persistent cryptosporidiosis (Table 5). On day 7 after exposure, mice that were supplemented with L. reuteri or L. acidophilus (Groups G and H, respectively) stripped less (p <0.05) oocysts of C. parvum compared to controls without exposure ( Group F). On day 14 after exposure, only mice supplemented with L. acidophilus (Group H) had lower fecal levels of C. parvum oocysts (p <0.05) compared to exposed controls (Group F); however, this level was not different (p> 0.05) from animals supplemented with L. reuteri (Group G). Unlike Exp. 1, intestinal infection of the ileal hairs was observed in exposed mice that had received the probiotic supplement. Less oocysts were detected in the hairs of animals supplemented with L. acidophilus (Group H) with no differences detected (p> 0.05) with respect to animals not exposed to C. parvum control. However, there were no differences (p> 0.05) with respect to the other exposed groups (Groups F and G). As in Exp. 1, no colonization of oocysts was observed in the proximal stomach or colon of mice.

TABLE 5

Effects of feeding with supplements of L. reuteri or L. acidophilus on oocyst shedding (log_ {10} oocysts / g ± EEM) in feces and colonization of the epithelium of the distal ileum (oocysts / 25 intestinal hairs ± SEM) of C578BL / 6 mice immunosuppressed by prior inoculation with MO-LP5 and exposed (+/-) to C. parvum Days after feeding probiotic Ileum Group 1 Day 13 2 Day 21 3 Day 28 4 Day 28 4 AND 0.00 ± 0.00 ** 0.00 ± pm 0.00 c ** 0.00 ± 0.00 c ** 0.00 ± pm 0.00 b F 0.00 ± 0.00 4.32 ± 0.17 a 3.88 ± 0.32 a 2.33 ± 0.81 a G 0.00 ± 0.00 2.96 ± 0.43 b 3.50 ± 0.29 a, b 2.13 ± 0.58 a H 0.00 ± 0.00 2.35 ± 0.62 b * 1.95 ± 0.13 b * 1.79 ± 0.71 a, b 1 Fifteen mice per group (5 mice per cage), unless otherwise indicated: * = 14, ** = 13. 2 Corresponds to a baseline sample taken just before exposure to C. parvum 3 Corresponds to day 7 after exposure to C. parvum 4 Corresponds to day 14 after exposure to C. parvum ^ a, b, c} Values within the same column with different superscript letters are different (p < 0.05).

Colonization by Lactobacillus

Faecal levels of Lactobacillus spp. Totals were similar among all treatments throughout the entire trial (Table 6). Statistical differences were found only for days 13 and 28. Fecal levels of L. reuteri were similar between baseline treatments, day 8 and day 13; however, the levels were approximately two orders of magnitude higher in Exp. 2 than baseline levels in Exp. 1. Generally, L. reuteri levels were high in all treatments with statistical differences observed for days 21 and 28 (see Table 6).

TABLE 6

Effects of feeding with supplements of L. reuteri or L. acidophilus on the fecal level of Lactobacillus spp. totals and L. reuteri (log 10 CFU / gEEM) of immunosuppressed C57BL / 6 mice by prior inoculation with MO-LP5 and exposed (+/-) to C. parvum Lactobacillus spp. Total - days after probiotic feeding Group 1 Day 1 Day 8 Day 13 Day 21 Day 28 AND 8.48 ± 0.18 8.54 ± pm 0.10 9.89 ± 0.15 a ** 9.58 ± pm 0.11 ** 9.91 ± 0.12 b F 8.43 ± 0.12 8.35 ± 0.15 8.80 ± 0.15 b 10.14 ± 0.11 10.66 ± 0.05 a G 8.48 ± 0.21 9.32 ± 0.10 8.50 ± 0.11 b 10.17 ± 0.12 10.72 ± 0.22 a H 8.94 ± 0.13 9.22 ± 0.09 9.71 ± 0.11 a ** 10.15 ± 0.20 * 10.94 ± 0.08 a L. reuteri AND 5.90 ± 0.44 6.58 ± 0.39 6.45 ± 0.58 ** 4.90 ± 0.56 b ** 8.09 \ pm 0.17 b F 6.44 ± 0.20 6.04 ± 0.47 4.72 ± 0.50 7.70 ± 0.32 a, b} 8.52 ± 0.13 b G 6.30 ± 0.36 7.76 ± 0.19 6.15 ± 0.39 7.79 ± 0.57 a 9.72 ± 0.11 a H 6.23 ± 0.43 8.58 ± 0.45 6.97 ± 0.49 ** 8.33 ± pm 0.18 a * 8.42 ± 0.57 a, b 1 Fifteen mice per group (5 mice per cage), unless otherwise indicated: * = 14, ** = 13. a, b Values within the same column with different letters of superscript are different (p <0.05)

Discussion - Exp. 2

The animals in this test were naturally colonized with L. reuteri ; however, the basal levels of L. reuteri were much higher than those described in Exp. 1. Due to this fact, the highest fecal level of L. reuteri cannot be correlated with the lowest oocyst shedding of C parvum (day 7). However, animals supplemented with L. reuteri stripped less oocysts (p <0.05) on day 7 after exposure to C. parvum than exposed control animals (Table 3).

Unfortunately, the faecal analysis for L. acidophilus was too difficult, therefore, the analysis was not completed. Mice supplemented with L. acidophilus stripped less oocysts (p <0.05) at 7 and 14 days after exposure to C. parvum compared to the exposed controls; however, this reduction was no different for animals supplemented with L. reuteri . This experiment suggests that L. acidophilus has a beneficial effect for the prevention of cryptosporidiosis similar to that of L. reuteri .

Conclusions

Together, prophylactic probiotic supplementation (specifically L. reuteri and L. acidophilus ) reduces the infectivity of C. parvum of a mammal in this murine model of AIDS, as evidenced by a reduction in oocyst shedding of oocysts. C. parvum in the stool. Knowing whether L. reuteri and L. acidophilus have a synergistic effect requires further study. These experiments have demonstrated a benefit of probiotics (specifically L. reuteri and L. acidophilus ) for the prevention of cryptosporidiosis in an immunosuppressed population. The level of L. reuteri fed to mice in our experiments was 1 x 10 8 CFU per mouse and per day. On a body weight basis, this would correspond to approximately 3 x 10 11 CFU per day for a 70 kilogram person. The present invention can be practiced by feeding at least, but not limited to 1 x 10 8 CFU per day per subject. The clinical evaluation of the use of L. reuteri to inhibit C. parvum in AIDS patients is anticipated in the near future.

The present invention, as supported by the experiments described above and the discussion thereof, is a medicament for the inhibition of infection of a mammal by Cryptosporidium parvum that is manufactured using enterally administered Lactobacillus reuteri in an amount that is therapeutically effective to inhibit such infection. That is, the present invention is a medicament for the inhibition of infection of the intestine of a mammal by the oocysts of Cryptosporidium parvum using Lactobacillus reuteri administered enterally in an amount that is therapeutically effective to inhibit said infection. In addition, the present invention is a medicament for increasing resistance to Cryptosporidium parvum infection in an immunocompromised mammal using Lactobacillus reuteri administered enterally in an amount that is therapeutically effective to inhibit such infection. The inhibition of infection of a mammal by Cryptosporidium parvum can be evidenced by a reduction in the oocyst shedding of Cryptosporidium parvum in the feces of the mammal being treated. Resistance to infection with Cryptosporidium parvum in an immunocompromised mammal can also be evidenced through a reduction in the oocyst shedding of Cryptosporidium parvum in the feces of the mammal being treated.

Although it is not easily evaluable in the murine studies described above, the enterally administered drug, which consists of Lactobacillus reuteri , also has the effect of reducing or relieving diarrhea (as measured through a reduction in the incidence of feces aqueous); especially in cases where the cause of such diarrhea is the presence of Cryptosporidium parvum and / or oocysts of Cryptosporidium parvum in the intestine.

Lactobacillus reuteri may be given enterally through any appropriate means, which includes but is not limited to:

(to)

the addition of Lactobacillus reuteri to a liquid through an osmotic pump and then enteral administration of the resulting liquid;

(b)

enteral administration through administration of an enteral capsule containing Lactobacillus reuteri ;

(c)

the consumption of a yogurt containing Lactobacillus reuteri at a concentration of at least 1.0 x 10 8 CFU of Lactobacillus reuteri / g;

(d)

the consumption of a dairy product containing Lactobacillus reuteri at a concentration of at least 1.0 x 10 8 CFU of Lactobacillus reuteri / ml;

(and)

the consumption of a seasoning containing 1.0 x 10 8 to 1.0 x 10 12 CFU of Lactobacillus reuteri / g, for example, by combining the seasoning with a meal and then consuming the food;

(F)

the consumption of a dosage unit comprising Lactobacillus reuteri and at least one excipient, for example, by combining the dosage unit with a food and then consuming the food, the dosage unit being for example a tablet, a capsule, a powder or liquid containing Lactobacillus reuteri ;

(g)

the consumption of a food product containing at least 1.0 x 10 8 CFU of Lactobacillus reuteri / g;

(h)

the consumption of a suspension containing Lactobacillus reuteri ;

(i)

enteral administration of a tablet containing Lactobacillus reuteri ; Y

(j)

the intake of an osmotic pump that will supply Lactobacillus reuteri to the intestinal tract.

A product that can be administered enterally that can be used in the practice of the present invention is a product that can be administered enterally that contains Lactobacillus reuteri in an amount that is therapeutically effective for inhibiting Cryptosporidium parvum infection of a mammal that has received it. Examples of such products include, but are not limited to:

(to)

a capsule containing Lactobacillus reuteri ;

(b)

a yogurt containing at least 1.0 x 10 8 CFU of Lactobacillus reuteri / g;

(c)

a dairy product containing at least 1.0 x 10 8 CFU of Lactobacillus reuteri / ml;

(d)

a condiment containing at least 1.0 x 10 8 CFU of Lactobacillus reuteri / g;

(and)

a tablet containing at least 1.0 x 10 8 CFU of Lactobacillus reuteri / g;

(F)

a suspension containing at least 1.0 x 10 8 CFU of Lactobacillus reuteri / ml;

(g)

a condiment containing Lactobacillus reuteri , said condiment being appropriate for combination with a food;

(h)

a dosage containing Lactobacillus reuteri with at least one excipient;

(i)

an osmotic pump that can be used to add Lactobacillus reuteri to a liquid;

(j)

an osmotic pump that can be consumed enterally and that supplies Lactobacillus reuteri directly to the gastrointestinal tract; Y

(k)

a food product that contains Lactobacillus reuteri .

Claims (19)

1. Use of Lactobacillus reuteri as a single organism in an amount that is therapeutically effective for inhibiting an infection in a mammal with Cryptosporidium parvum for the production of an enteral drug for the inhibition of said infection. 2. Use of Lactobacillus reuteri according to claim 1 for the production of an enteral drug to inhibit an infection in a mammal by Cryptosporidium parvum , in which Lactobacillus reuteri is used in an amount that is therapeutically effective to inhibit said infection as evidenced by through a reduction in the oocyst shedding of Cryptosporidium parvum in feces. 3. Use of Lactobacillus reuteri according to claim 1 for the production of an enteral drug to inhibit an infection of the intestine of a mammal by the oocysts of Cryptosporidium parvum , in which Lactobacillus reuteri is used in an amount that is therapeutically effective to inhibit said infection. . 4. Use of Lactobacillus reuteri according to claim 3 for the production of an enteral drug to inhibit infection of the intestine of a mammal by Cryptosporidium parvum , in which Lactobacillus reuteri is used in an amount that is therapeutically effective to inhibit said infection as is evidence through a reduction in the oocyst shedding of Cryptosporidium parvum in feces. 5. Use of Lactobacillus reuteri according to claim 1 for the production of an enteral drug to increase resistance to infection by Cryptosporidium parvum in an immunocompromised mammal, in which Lactobacillus reuteri is used in an amount that is therapeutically effective to inhibit said infection. 6. Use of Lactobacillus reuteri according to claim 5 for the production of an enteral drug to increase resistance to Cryptosporidium parvum infection in an immunocompromised mammal, in which Lactobacillus reuteri is used in an amount that is therapeutically effective to inhibit such infection. as evidenced by a reduction in the oocyst shedding of Cryptosporidium parvum in feces. 7. Use of Lactobacillus reuteri according to any of claims 1-6 for the production of a pharmaceutical enteral liquid to which Lactobacillus reuteri has been added through an osmotic pump. 8. Use of Lactobacillus reuteri according to any of claims 1-6 for the production of a pharmaceutical enteral capsule containing Lactobacillus reuteri . 9. Use of Lactobacillus reuteri according to any of claims 1-6 for the production of an enteral yogurt containing Lactobacillus reuteri at a concentration of at least 1.0 * 10 8 CFU of Lactobacillus reuteri / ml. 10. Use of Lactobacillus reuteri according to any of claims 1-6 for the production of an enteral dairy product containing Lactobacillus reuteri at a concentration of at least 1.0 * 10 8 CFU of Lactobacillus reuteri / ml. 11. Use of Lactobacillus reuteri according to any of claims 1-6 for the production of an enteral condiment containing Lactobacillus reuteri at a concentration of at least 1.0 * 10 8 CFU of Lactobacillus reuteri / g. 12. Use of Lactobacillus reuteri according to claim 11, wherein the seasoning is combined with a food before consumption thereof. 13. Use of Lactobacillus reuteri according to any of claims 1-6 for the production of an enteral dosage unit containing Lactobacillus reuteri and at least one excipient. 14. Use of Lactobacillus reuteri according to claim 13, wherein the dosage unit is combined with the food. 15. Use of Lactobacillus reuteri according to any of claims 1-6 wherein the pharmaceutical product is a food product containing at least 10 8 CFU of Lactobacillus reuteri / g. 16. Use of Lactobacillus reuteri according to any of claims 1-6, wherein the enteral pharmaceutical product is a suspension containing Lactobacillus reuteri . 17. Use of Lactobacillus reuteri according to any of claims 1-6, wherein the enteral pharmaceutical product is a tablet containing Lactobacillus reuteri . 18. Use of Lactobacillus reuteri according to any of claims 1-6, wherein the enteral pharmaceutical product is an ingestible osmotic pump that can provide Lactobacillus reuteri to the gastrointestinal tract.

         \ newpage
      

19. Use of Lactobacillus reuteri according to claim 1 for the production of a drug to reduce the incidence in a mammal of diarrhea caused by the presence of Cryptosporidium parvum in the intestine.